The PCR techniques are generally described in U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188. The PCR technique generally involves a process for amplifying any desired specific nucleic acid sequence contained within a nucleic acid molecule. The PCR process includes treating separate complementary strains of the nucleic acid with an excess of two oligonucleotide primers. The primers are extended to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The PCR process is carried out in a simultaneous step-wise fashion and can be repeated as often as desired in order to achieve increased levels of amplification of the desired nucleic acid sequence. According to the PCR process, the sequence of DNA between the primers on the respective DNA strains are amplified selectively over the remaining portions of the DNA and selected sample. The PCR process provides for the specific amplification of a desired region of DNA.
The method of the present invention uses the PCR amplification process that allows simultaneous amplification of a “target gene”, a “housekeeping” gene and competitive templates for each of these genes. According to the present invention, the terms “target DNA sequence” and “target gene” generally refer to a gene of interest for which there is a desire to selectively amplify that gene or DNA sequence. The term “housekeeping” gene refers to genes that are suitable as internal standards for amount of RNA per PCR reaction. In a general and overall sense, a key to the present invention is the simultaneous use of primers for target genes, primers for a housekeeping gene, and two internal standard competitive templates comprising mutants of the target genes and housekeeping gene. These mutations can be point mutations, insertions, deletions or the like.
There is a need for quantitative measurement of gene expression which controls for the expression of all relevant genes that may be involved in individuals at risk for certain diseases, including, for example, bronchogenic carcinoma. The present invention addresses these needs by providing a method for gene expression measurement by quantitative RT-PCR that allows simultaneous expression measurement of many genes. The multiplex competitive reverse transcriptase-poly erase chain reaction is generally described in the Willey and Willey et al. U.S. Pat. Nos. 5,639,606; 5,643,765 and 5,876,978 which are fully incorporated herein by reference, along with all other references disclosed herein and listed at the end of the specification. According to one aspect of the present invention, the mRNA expression of mGST, GSTM3, GSTT1, GSTP1, GSHPx and GSHPxA and the combined expression of GSTM1, 2, 4, 5 are simultaneously measured in the primary NBECs of non-lung cancer patients, primary NBECs from lung cancer patients, and in cultured NBECs from non-lung cancer patients.
Normal bronchial epithelial cells (NBECs) are at an increased risk for oxidative damage following inhalational exposure to reactive oxygen species in cigarette smoke (1, 2), ozone (3), possibly asbestos (4), and other particulates in the environment. NBECs also are exposed to endogenous oxidative products produced through normal cellular metabolism (5) and during inflammation (6, 7). In addition, inhaled daughters of radon-2222 decay (polonium218 and polonium214) may deposit on NBECs and emit α particles that generate reactive oxygen products as they encounter the cells. NBECs also are exposed through inhaled cigarette smoke or urban air pollution to polycyclic aromatic hydrocarbons (PAHs). These procarcinogens may be metabolically activated in the cytoplasm and subsequently damage nuclear DNA. Damage to NBECs and adjacent structures from oxidants and or activated carcinogens may result in a variety of pulmonary disorders, including bronchogenic carcinoma, pulmonary fibrosis, chronic bronchitis, and emphysema (5, 8).
NBECs express several enzymes, including glutathione-S-transferase (GSTs) and glutathione peroxidases, that are capable of preventing or reducing injury from reactive oxidants or carcinogens. The GST enzymes conjugate reactive chemical groups, including reactive oxygen species and diol-epoxide ultimate carcinogens, to glutathione and thereby prevent them from binding to and damaging DNA (9). There are several classes of GSTs, including one microsomal class (mGST) and four cytosolic classes: GSTA, GSTM, GSTP, and GSTT (10, 11). In addition, a human homologue of rat GSTK1 has been reported (12). Each GST enzyme has substrate specificity, but there is considerable overlap (13). For example, diol-epoxides derive from PAH procarcinogens are metabolized by GSTP1 and GSTM1-3 (14). Other substrates for the cytosolic GSTs include steroids, alkenals, and quinones (9). In contrast to the cytosolic GST enzymes, mGST has very little specificity for epoxides (15). However, mGST has activity against a broad range of other substrates, including styrene-7-8-oxide (16), 1-chloro-2,4-dinitrobenzene, and cumene hydroperoxide (17). Further, various halogenated alkynes and alkenes are metabolized preferentially by mGST compared to the cytosolic forms (13, 18).
The glutathione peroxidase enzymes catalyze the inactivation of peroxides (including hydrogen peroxide and lipid peroxides) using reduced glutathione as a cofactor (19). Several enzymes have glutathione peroxidase activity, including GSHPx (19), GSHPxA (a secreted form; Ref 20), mGST (21), GSTA (22), and GSTM3 (23).
Both intertissue and interindividual variation in the expression of GST and glutathione peroxidase genes have been reported (14, 24-27). In addition, the expression of some GST and glutathione peroxidase genes is altered in carcinoma tissues (14, 20, 24, 25, 28, 29). Because there is intertissue variation in the expression of these genes, it is important to measure expression specifically in the progenitor cell for bronchogenic carcinoma, the bronchial epithelial cell. There is very little information presently available regarding quantitative levels of GST or glutathione peroxidase gene expression in primary NBECs relative to primary bronchogenic carcinoma tissue.
The inventors herein have discovered that interindividual variation in GST enzyme gene expression translates into variation in risk for bronchogenic carcinoma. For example, in some epidemiological studies, GSTM1 null individuals have an increased risk (30, 31). However, the results of other studies are contradictory (32). One hypothesis to explain these different results is that because the multiple GST and glutathione peroxidase enzymes have a broad substrate overlap, a decrease in the expression level of one GST or glutathione peroxidase may be compensated for by increased expression of another. Thus, the expression patterns for multiple relevant GST and glutathione peroxidase enzymes may be more closely associated with risk than the expression of each individual gene. Consequently, studies that do not control for expression of all relevant genes may generate data that are difficult to interpret.